The present invention relates to plasmids derived from ammonia oxidizing bacteria and use of the same.
Ammonia oxidizing bacteria belonging to chemoautotrophic bacteria are microorganisms that obtain energy necessary for their life action only by biological reaction for oxidizing ammonia into nitrous acid. The ammonia oxidizing bacteria have an important role in the nitrogen cycle in nature. In addition, they are also important as microorganisms mediating the nitration process as the main reaction of nitrogen elimination in the biological treatment of human wastewater, domestic wastewater and industrial wastewater by the activated sludge process. In recent years, there has been a tendency to require an improvement in the efficiency of nitrogen elimination in the above biological treatment of wastewater from a viewpoint for the prevention of eutrophication in waters, and the function of ammonia oxidizing bacteria has drawn much attention. However, ammonia oxidizing bacteria exhibit extremely low speed in the oxidation of ammonia and in the growth because they cannot obtain energy by oxidative degradation of organic matter in contrast to the ordinary aerobic heterotrophic microorganisms. In addition, ammonia oxidizing bacteria are susceptible to the inhibition of growth by various organic chemical substances or metal elements, so that the degree of nitrogen elimination can easily be lowered when harmful matter is mixed in the influent raw wastewater during the biological treatment of wastewater.
As a means of overcoming the above defects of ammonia oxidizing bacteria, genetic recombination technique may be used, which is a technique of transferring an appropriate foreign gene fragment to ammonia oxidizing bacteria for improving the function of genes that code for enzymes associated with the ammonia oxidation and that exist on the chromosomes of ammonia oxidizing bacteria, followed by expression of these fragments, or transferring enzyme genes to ammonia oxidizing bacteria, which genes code for enzymes exhibiting detoxifying action against harmful substances to the ammonia oxidizing bacteria, followed by expression of these genes. No vector system, however, has been established so far for which ammonia oxidizing bacteria are used as a host organism; therefore, such genetic recombination technique cannot be carried out. There has been a great demand for the development of vector systems for which ammonia oxidizing bacteria are used as a host organism.
Under these circumstances, the present inventors have extensively studied. As a result, they have found out plasmids replicable in the cells of ammonia oxidizing bacteria, thereby completing the present invention.
Thus, the present invention provides:
1) A plasmid characterized by having the nucleotide sequence of SEQ ID NO: 1 (which plasmid is hereinafter referred to as the present plasmid S).
2) A plasmid characterized by comprising about 1.8 kilobase pairs and being represented by the restriction map shown in FIG. 1.
3) A plasmid characterized by having the nucleotide sequence of SEQ ID NO: 2 (which plasmid is hereinafter referred to as the present plasmid L).
4) A plasmid characterized by comprising about 1.9 kilobase pairs and being represented by the restriction map shown in FIG. 2.
5) A plasmid characterized by having at least a part of the nucleotide sequences of SEQ ID NOs: 1 and 2 and further containing a selective marker gene (which plasmid is hereinafter referred to as the present chimeric plasmid).
6) The plasmid according to item 5, characterized in that it is replicable in cells of either ammonia oxidizing bacteria or Esherichia coli. 
7) A plasmid characterized by comprising about 7.6 kilobase pairs and being represented by the restriction map shown in FIG. 3.
8) A transformant characterized in that it is obtained by transferring at least one plasmid according to any one of items 1 to 7 into a host organism.
9) A method for detecting ammonium ions in an aqueous solution, characterized in that a transformant obtained by transferring into ammonia oxidizing bacteria a plasmid according to any one of items 1 to 7, into which a luminescent organism derived gene coding for a protein associated with bioluminescence has been inserted, is brought into contact with both a sample aqueous solution and a substrate for the bioluminescence, and the bioluminescence of the said transformant is measured.
10) A method for preparing ammonia oxidation bacteria having resistance to nitrification inhibitory substances, characterized in that a plasmid according to any one of items 1 to 7, into which a gene coding for an enzyme that catabolizes nitrification inhibitory substances has been inserted, is transferred into ammonia oxidation bacteria to obtain a transformant.
11) A method for enhancing the ability of ammonia oxidizing bacteria to oxidize ammonia, characterized in that a plasmid according to any one of items 1 to 7, into which a gene coding for an enzyme associated with ammonia oxidation has been inserted, is transferred into ammonia oxidation bacteria to obtain a transformant.